Interference of hemoglobin variants in HbA1c quantification.

Fasting blood glucose and glycated hemoglobin (HbA1c) are routine biomarkers to screen and monitor diabetes mellitus. HbA1c results from glycation at the N-terminus of the ß globin chain of tetrameric human hemoglobin. Fasting blood glucose level varies with the nature and amount of food intake, physical exercise, etc., and, accordingly, is a short-term measure of glucose control. In contrast, HbA1c provides an average measure of glucose control for the long-term (8-12 weeks). Unfortunately, genetic variants of hemoglobin may interfere with HbA1c quantification using ion exchange chromatography, capillary electrophoresis, immunoassay and boronate affinity chromatography. Mass spectrometry, however, measures total glycation of hemoglobin across both α and ß globin chains and correlates well with the ion exchange based method. Additionally, mass spectrometry based quantification is not impacted by the presence of genetic variants of hemoglobin and thus might be a better analytical choice for diabetes mellitus.

Effect of an ionic liquid on the unfolding of human serum albumin: a fluorescence correlation spectroscopy study.

The effect of the room temperature ionic liquid (RTIL) 1-pentyl-3-methyl-imidazolium bromide ([pmim][Br]) on the unfolding of a protein, human serum albumin (HSA), is studied by fluorescence correlation spectroscopy (FCS). The structural fluctuations of the protein exhibit three characteristic time constants, namely, ~3, ~35 and ~260 µs. On addition of the RTIL, the dynamics become slightly slower, with time constants of ~5, ~40 and ~350 µs. The two fast components (3 and 35 µs in the absence of RTIL and 5 and 40 µs in the presence of RTIL) are assigned to chain motion of the protein. The slowest component (260 or 350 µs) may arise from detachment (unbinding) of the non-covalent dye from the protein. In the absence of RTIL--and on addition of guanidinium hydrochloride (GdnHCl)--as the protein unfolds, the contribution of the fastest component increases rapidly from 10% at 1 M to 40% at 6 M, and its time constant decreases from 3 µs to 1 µs. In the presence of RTIL, the addition of GdnHCl causes significant changes in both the structure (CD spectrum) and the time constants of conformational fluctuation. In the presence of the RTIL, the addition of GdnHCl gives rise to a very slow component (1025 µs in 1 M and 560 µs in 6 M GdnHCl). It is proposed that the guanidinium cation (GdnH(+)) repels the imidazolium cation ([pmim](+)) at the protein surface, and this causes a change in the structure and dynamics of the protein. On addition of 6 M GdnHCl, the diffusion coefficient of C153 bound to HSA decreases. The hydrodynamic radius of the denatured protein (in 6 M GdnHCl) is larger than that of the native protein (about 1.75 times in the absence of RTIL and 2.6 times in the presence of RTIL).